The present invention relates to live attenuated RTX-producing bacteria of the family Pasteurellaceae, methods for the production of such bacteria, to vaccines comprising such bacteria, methods for the production of such vaccines and methods for the protection of man and animals against infection with virulent RTX-producing bacteria of the family Pasteurellaceae.
The family of Pasteurellaceae comprises the genera Haemophilus, Actinobacillus and Pasteurella. The bacteria of this family are also often referred to as bacteria of the HAP-group. Several species of these closely related genera are known to express homologous calcium-dependent, pore-forming cytotoxins, the so-called RTX toxins. (RTX stands for repeat in toxin). RTX toxin-producing bacteria of this family are the cause of a whole range of infectious diseases, influencing both man and animals.
RTX toxins are also known from other genera, not related to the HAP-group, such as Escherichia and Bordetella. These RTX toxins in some respects resemble the RTX-toxins of the HAP-group.
The RTX toxins have been extensively reviewed by Braun et al. (Critical Rev. in Microbiol. 18(2): 115-158 (1991) Gram-negative strains have also been reviewed in Welch, R. A. (Molecular Microbiology 5/3: 521-528 (1991)) and in Welch et al. (Inf. Agents and Disease 4: 254-272 (1995)).
It is the presence of the RTX toxins in the RTX-producing members of the Pasteurellaceae family of bacteria, that highly contributes to the pathogenic character of these bacteria for both man and animals.
All RTX toxins display some kind of cytotoxic or cytolytic activity. The target-cell- and host-specificity differ however, depending on the toxin and on differences in acylation (McWhinney et al.; J. Bact. 174: 291-297 (1992) and Hackett et al.; J. Biol. Chem. 270: 20250-20253 (1995)). As a result of the difference in target cells, the various toxins of the RTX toxin family are known e.g. as haemolysin, cytolysin or cytotoxin.
Although many RTX-producing members of the HAP-group are known, some of them are notorious for the economic damage they cause.
Actinobacillus pleuropneumoniae produces RTX toxins that are cytotoxic/cytolytic to pig, horse, bovine and human erythrocytes, to rabbit and porcine neutrophils and to porcine alveolar macrophages. (Rosendal et al; Am. J. Vet. Res. 49: 1053-1058 (1988), Maudsley J. R. and Kadis S; Can. J. Microbiol. 32: 801-805 (1986), Frey. J and Nicolet. J; Inf. and Imm. 56:2570-2575 (1988), Bendixon et al; Inf. and Imm. 33: 673-676 (1981), Kamp, E. M. and van Leengoed, L. A. M. G.; J. Clin. Microbiol. 27: 1187-1191 (1989)).
Actinobacillus infection in pigs causes severe economic losses to pig industry, due to acute mortality in young pigs and reduced weight gain in older animals.
The Pasteurella haemolytica RTX toxin activity is mainly directed against neutrophils and monocytes/macrophages from ruminants (Shewen and Wilie; Inf. and Immun. 35, 91-94 (1982), Baluyut et al.; Am. J. Vet. Res. 42: 1920-1926 (1981), Himmel et al.; Am. J. Vet. Res. 43: 764-767 (1982)).
Pasteurella infections cause severe problems in ruminants, especially cattle and sheep.
Mastitis and pneumonia are seen in both sheep and cattle, whereas Shipping Fever causes additional problems in cattle. Economic losses due to Pasteurella infections are high. Other, non-HAP-group bacteria are also known to produce RTX toxins.
The E. coli haemolysin is toxic for a large variety of cells, from a large number of different animal species. It lyses erythrocytes from many animal species within a few minutes after contact. (Cavalieri, S. J. and Snyder, I. S.; Inf. and Imm. 37: 966-974 (1982), Gadeberg et al; Inf. and Imm. 41: 358-364 (1983), Keane et al; Am. J. Pathol. 126:305-357 (1987), Bhadki et al; J. Exp. Med. 169: 737-754 (1989)).
The Bordetella pertussis haemolysin also displays a large host-cell range. (Shattuck, R. L. and Storm, D. R.; Biochemistry 24: 6323-6328 (1985), Hewlett et al, In Protein Bacterial Toxins, Rappuoli, R. et al. (Eds), Stuttgart, Fisher-Verlag 249-257 (1990)).
The genetic organisation of the operons involved in the synthesis, activation and transportation of the RTX toxins in Gram-negative bacteria has been reviewed recently by Coote, J. G. (FEMS Microbiology reviews 88: 137-162 (1992)) In general, the RTX operon contains four genes: the actual Toxin gene (A), an Activator gene (C), and two genes (B and D (and E in Bordetella pertussis)) encoding proteins involved in secretion of the toxin into the surrounding medium. The primary translation product of the Toxin-gene (A) is a non-toxic protein.
The role of the Activator gene (C) is of paramount importance in that the gene product encoded by this gene activates the toxic activity of the RTX toxin by posttranslational modification.
This activation results in a structural modification of the toxin. In e.g. Bordetella pertussis, the posttranslational no modification of the RTX toxin is caused by amide-linked palmitoylation of a lysine residue (Hackett et al.; Science 266: 433-435 (1994). The RTX toxin of E. coli could be activated in vitro by transfer of a fatty acyl group from acyl carrier protein to prohaemolysin (Issartel et al.; Nature 351: 759-761 (1991)).
It is known (see e.g. Coote, J. G.; FEMS Microbiology reviews 88: 137-162 (1992)), that RTX toxins are important virulence factors in bacteria belonging to the Pasteurellaceae. This has been shown for e.g. Actinobacillus pleuropneumoniae by Tascon et al.(Mol. Microbiol. 14: 207-216 (1994)) and by Jansen et al. (Inf. and Imm. 63: 27-37 (1995)).
Virulence factors are known to be the main targets for incorporation in vaccines.
Therefore, several attempts have been made to use RTX toxins as subunit vaccines.
In vivo synthesised RTX toxins of the HAP-group are per se produced in the presence of the RTX Activator protein. Therefore, RTX toxins are always posttranslationally modified into highly toxic proteins.
Given their high toxicity it is clear that the RTX toxins need to be detoxified before they can be used as a vaccine component.
Subunit vaccines based on in vivo synthesised RTX toxins from A. pleuropneumoniae that lost their toxicity have been described earlier, e.g. in European Patent EP No. 0.354.628, in which subunit vaccines based upon a haemolysin and a cytotoxin of A. pleuropneumoniae are disclosed, and in European Patent EP No 0.453.024, in which A. pleuropneumoniae subunit vaccines based upon haemolysins, cytotoxins and outer membrane proteins are disclosed.
Subunit vaccines based on RTX toxins from Pasteurella haemolytica have also been disclosed, e.g. in U.S. Pat. No. 5,055,400, Canadian Pat. Appl. CA 2,014,033 and Canadian Pat. Appl. CA 2,081,950.
RTX toxins as subunits for use in vaccines are easily obtained from the supernatant of bacterial cultures of the wild-type strains. Another way of obtaining the RTX toxin as a subunit has been proposed in Canadian Patent Application CA 2,045,950, in which heterologous expression of the genes encoding the A. pleuropneumoniae RTX-protein in the heterologous bacterial strain E. coli has been described. No vaccine experiments with the RTX toxins so obtained were shown however.
A comparable approach for the production of subunit vaccines has been proposed in European Patent EP 0.500.736. In this patent, the sequence of the RTX Toxin gene (A) and an Activator gene (C), is disclosed. Also a heterologous expression system for the expression of the Toxin gene A in the presence or absence of the Activator gene C is disclosed. No vaccination experiments with the toxin subunit were however, described.
There are however, three important disadvantages to all RTX toxin subunit vaccines:
high amounts of antigenic material are needed in order to adequately trigger the immune system.
usually, only B-cell immunity is triggered.
a live pathogenic bacterium has many important immunogenic molecules, such as Outer Membrane Proteins and capsular polysaccharides, all being important for protection. Therefore, in order to produce an efficient subunit vaccine, one must additionally include as many other immunogenically important antigens as possible.
Next to the obvious problems mentioned under points one and two, especially the third point makes it difficult to make an efficient subunit vaccine.
This is e.g. illustrated by the A. pleuropneumoniae subunit vaccine disclosed in European Patent EP No 0.453.024 mentioned above, in which four different subunits (three RTX toxins and an outer membrane protein) are combined in one vaccine.
It is clear that in order to overcome the disadvantages of subunit vaccines against Pasteurellaceae infection, a live attenuated vaccine would be highly desirable.
A live attenuated vaccine has the following advantages:
it can be administered in low doses (it is self-replicating)
it closely mimics the natural/wild-type infection
it provides all the possible immunologically important antigens at the same time.
Nevertheless, in spite of the clear advantages, live vaccines based on bacteria of the HAP-group producing a less active RTX toxin were not available prior to the present invention. The reason for the lack of live attenuated vaccines is clearly illustrated by the following paradox:
The first characteristic of a live attenuated vaccine strain is that it should not produce active RTX toxin, since as mentioned above, it is this RTX toxin that makes strains of the HAP-group so virulent.
A live attenuated bacterium attenuated through the inability to express RTX toxins would, however, per se lack the most important virulence factor i.e. the RTX toxins, and will therefore not trigger an immune response against this toxin.
As a consequence, if the RTX gene(s) is (are) deleted from strains of the HAP-group and such attenuated strains are used as a basis for a vaccine against diseases caused by virulent wild-type strains of the HAP-group, only partial protection is achieved: one would never obtain protective immunity against the most important virulence factor of these wild-type strains i.e. the RTX toxin.
Therefore, vaccines based on bacteria with a deletion of the RTX toxin cannot possibly be expected to provide protection against the damaging effects of the RTX-toxin after infection with wild-type strains.
Strains lacking the apxI operon were made i.a. by Reimer et al. (Microbial Pathog. 18: 197-209 (1995)), who deleted all genes playing a role in the synthesis and transportation of A. pleuropneumoniae ApxI Toxin.
Such strains are non-virulent as expected, since they do no longer excrete the most important virulence factor the RTX toxin; but as a consequence no antibodies, let alone protective antibodies, will be induced against the RTX toxins.
The present application for the first time provides live attenuated RTX toxin-producing bacteria of the family Pasteurellaceae, that do produce the RTX-A toxin, but in a non-activated form.
These bacteria have as a remarkable feature that they are on the one hand attenuated, whereas on the other hand, they are still capable of producing the RTX toxin.
This is achieved by modifying the bacteria in such a way that they do not produce a functional RTX activator protein. Expression of the RTX-A toxin, however, is not impaired.
The advantage of live attenuated strains according to the present invention over subunit vaccines as well as over live strains from which the RTX toxin genes are deleted is that:
they do produce the RTX toxin so that protective antibodies against this toxin are induced
they nevertheless are attenuated in their virulence since they produce the RTX toxin in a non-toxic form
they additionally possess all the other antigens that next to the RTX toxin are necessary to obtain an efficient immune response.
RTX-A toxin in a non-activated form is considered to be non-toxic, i.e. not having the same toxic effect as the activated toxin. As mentioned above, this is achieved by modifying the bacteria in such a way that they do not produce a functional RTX activator protein. Expression of the RTX-A toxin, however, is not impaired.
A functional RTX activator protein is considered to be a protein that has all the characteristics of the RTX activator protein as expressed in a wild-type bacterium, and is expressed at the wild-type level.
Therefore, a non-functional RTX activator protein is considered to be a protein that lacks some or all of the characteristics of the RTX activator protein as expressed in a wild-type bacterium, and/or is expressed at a level, insufficient to obtain wild-type levels of activated RTX toxin.
The following must be stressed here: if the non-functional RTX activator protein lacks all of the characteristics of the RTX activator protein as expressed in a wild-type bacterium, the bacterium will produce no activated RTX toxin at all. If however the non-functional RTX activator protein only lacks some of the characteristics of the RTX-activator protein as expressed in a wild-type bacterium, the bacterium may produce part of the RTX toxin in an activated form and part of the RTX toxin in a non-activated form. This is e.g. the case if due to a mutation the Activator protein is expressed, but the activation efficiency of the Activator protein is reduced. The activation speed is the speed with which the Activator protein activates the RTX toxin, i.e. converts the RTX toxin from its non-activated form to its activated form.
It thus goes without saying that bacteria that produce part of the RTX toxin in a non-activated form and part in an activated form are also embodied in the present invention.
Inability to obtain wild-type levels of activated RTX toxin may be the result of a decreased activity of the RTX activator protein. It may also be the result of a decreased expression level of the RTX activator protein, or a combination of the two possibilities.
As a consequence, RTX activator proteins with a decreased activity and/or a decreased expression level are within the scope of the invention.
Alternatively, it is possible to modify the target-site of the RTX Activator protein, i.e. the acylation-site at the RTX-toxin. If this site is modified to the extent that acylation is decreased or absent, this also results in the production of an RTX-toxin in a non-activated form. The acylation site can easily be mutated using recombinant DNA techniques. Mutation can e.g. be obtained by deletion of a restriction fragment that comprises the acylation site, or by site-directed mutagenesis of the acylation site.
A live attenuated bacterium with a non-functional RTX activator protein can be obtained in several ways. One possibility is to introduce a mutation into the gene encoding the RTX-activator protein, preferably by utilising recombinant DNA techniques.
A mutation is understood to be a change of the genetic information in the above-mentioned region with respect to the genetic information present in this region of the genome of the wild-type bacterium. The mutation is, for example, a nucleic acid substitution, deletion, insertion or inversion, or a combination thereof resulting in a bacterium which fails to produce a functional RTX activator protein.
Much is currently known about the location, restriction pattern and often even the nucleotide sequence of the RTX activator genes of RTX toxin producing strains of the HAP-group. This information can e.g. be found in the review by Coote, J. G. (FEMS Microbiology reviews 88: 137-162 (1992)), who gives an overview of structural and functional relationship between the various RTX toxins. Very detailed information about specific RTX toxins can be found in e.g. U.S. Pat. No. 5,055,400, that refers to the RTX-gene of Pasteurella haemolytica, and in Frey et al.; J. Gen. Microbiol. 139: 1723-1728 (1993) and Frey et al.; Proceedings of the HAP-conference U.K., Edinburgh 1994, concerning all genes playing a role in the synthesis and transportation of A. pleuropneumoniae RTX toxins.
Mutation of the gene encoding the RTX activator protein or of sequences involved in the transcription/translation of that gene can be obtained in several ways. One possibility is cloning of the relevant sequences of the RTX activator gene in a vector, excision of part or all of the RTX sequences using restriction enzymes and replacement of the wild-type RTX toxin gene with the mutated sequences. Such a replacement is e.g. performed by the well-known technique of homologous recombination.
Another possibility is the use of site-directed mutagenesis, to obtain the desired mutation.
These standard recombinant DNA techniques are described e.g. by Sambrook et al. in Molecular Cloning: a laboratory manual Cold Spring Harbor Laboratory Press (1989)
Thus, in a preferred embodiment, the bacterium has a mutation in the gene encoding the RTX-activator protein. This mutation may lead to a less active or fully inactive RTX-activator protein depending on the size and character of the mutation.
In a more preferred form, the mutation in the gene encoding the RTX activator protein is a deletion. The deletion may vary highly in size: it may e.g. be as small as one nucleotide, causing frame-shift. On the other hand, the whole gene encoding the RTX activator protein may be deleted.
Another possibility is to leave the gene encoding the RTX-activator protein intact, but to decrease the expression level of the RTX activator protein.
Since the Toxin gene and the Activator gene are transcribed from the same promoter in a polycistronic messenger RNA, it is not possible to decrease the transcription level without concomitantly decreasing the level of expression of the RTX toxin.
However, modification of the expression level of the RTX activator protein can be achieved by introducing a mutation into the ribosome binding site upstream of the gene encoding the RTX activator protein, preferably by utilising recombinant DNA techniques.
Therefore, in another preferred embodiment, the bacterium has a mutation in the region controlling the translation of the RTX activator mRNA, such as the ribosome binding site. Such a mutation influences the efficiency of translation of the RNA encoding the RTX activator protein.
Ribosome binding sites are in general easily detected on the basis of their consensus-motive and the relative distance of about 5-6 nucleotides between the ribosome binding site and the start codon. In many cases, e.g. for several RTX activator genes of A. pleuropneumoniae they are published (Frey et al. Gene 142: 97-102 (1994)).
In a more preferred form of this embodiment, the mutation in the region controlling the translation of the RTX activator mRNA is a deletion. The deletion may e.g. comprise a deletion of one or more nucleotides of the ribosome binding site.
Still another possibility to obtain a live attenuated bacterium with a non-functional RTX activator protein is to add a nucleic acid sequence that codes for an antisense RNA, that can bind to the messenger RNA encoding the Activator protein. Expression of such a sequence then leads to a decrease in the level of activator protein.
Antisense RNA is RNA that has a sequence that is partially or fully complementary to the sequence of the messenger RNA (mRNA) to which it is antisense.
In the most preferred embodiment, the live attenuated bacterium according to the present invention is Actinobacillus pleuropneumoniae. 
The present invention also relates to vaccines for the protection of animals against infection with an RTX toxin producing bacterium of the family Pasteurellaceae.
Such vaccines are based on a live attenuated RTX toxin producing bacterium according to the invention and a pharmaceutically acceptable carrier.
These vaccines comprise at least an immunogenically effective amount of the live attenuated RTX toxin producing bacterium according to the invention. Immunogenically effective means that the amount of live attenuated RTX toxin producing bacterium administered at vaccination is sufficient to induce in the host an effective immune response to virulent forms of the RTX toxin producing bacterium.
The useful dosage to be administered will vary depending on the age, weight and mammal vaccinated, the mode of administration and the type of pathogen against which vaccination is sought.
The vaccine may comprise any dose of bacteria, sufficient to evoke an immune response. Doses ranging between 103 and 1010 bacteria are e.g. very suitable doses.
In addition to an immunogenically effective amount of the live attenuated RTX toxin producing bacterium described above, a vaccine according to the present invention also contains a pharmaceutically acceptable carrier.
Such a carrier may be as simple as water, but it may e.g. also comprise culture fluid in which the bacteria were cultured. Another suitable carrier is e.g. a solution of physiological salt concentration.
Other examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilisers such as SPGA, carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein containing agents such as bovine serum or skimmed milk and buffers (e.g. phosphate buffer).
Optionally, one or more compounds having adjuvant activity may be added to the vaccine. Adjuvantia are non-specific stimulators of the immune system. They enhance the immune response of the host to the invading pathogen. Examples of adjuvantia known in the art are Freunds Complete and Incomplete adjuvans, vitamin E, non-ionic block polymers, muramyldipeptides, ISCOMs (immune stimulating complexes, cf. for instance European Patent EP 109942), Saponins, mineral oil, vegetable oil, and Carbopol (a homopolymer).
Adjuvantia, specially suitable for mucosal application are e.g. the E. coli heat-labile toxin (LT) or Cholera toxin (CT). Other suitable adjuvants are for example aluminium hydroxide, phosphate or oxide, oil-emulsions (e.g. of Bayol F(copyright) or Marcol 52(copyright), saponins or vitamin-E solubilisate.
Therefore, in a preferred form, the vaccines according to the present invention comprise an adjuvant.
For administration to animals, the vaccine according to the presentation can be given inter alia intranasally, intradermally, subcutaneously, by aerosol or intramuscularly.
In a more preferred embodiment, the vaccine according to the present invention additionally comprises one or more antigens selected from other pathogenic microorganisms or viruses. Such a vaccine can be obtained by adding one or more antigens selected from other pathogenic bacteria or viruses to the live attenuated RTX toxin producing bacterium according to the invention and a pharmaceutically acceptable carrier as described above.
Of course, it is possible to add not only one or more antigens, but also one or more of the whole pathogens as such, in an inactivated or live form.
It can alternatively be obtained by cloning the genetic information encoding one or more antigens selected from other pathogenic microorganisms or viruses into the live attenuated RTX toxin producing bacterium, using known recombinant DNA technology. Bacteria according to the present invention are very suitable as carriers, i.e. vectors, for such genetic information, due to their attenuated character. Vaccines based on bacteria according to the present invention that additionally carry genetic information encoding one or more antigens selected from other pathogenic microorganisms or viruses are capable of immunising against two or more diseases at the same time. This is of course less stressing for the animal to be vaccinated than separate vaccinations with each of the pathogens, both from a medical and a physical point of view.
In an even more preferred embodiment, the vaccine according to the present invention comprises live attenuated RTX toxin producing bacterium belonging to the species Actinobacillus pleuropneumoniae. 
In a still even more preferred form, these antigens are selected from, but not limited, to the group consisting of Porcine Reproductive Respiratory Syndrome (PRRS) virus, Pseudorabies virus, Porcine Influenza virus, Porcine Parvovirus, Transmissible Gastroenteritis virus, rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Streptococcus suis. 
In another form of the even more preferred embodiment, the vaccine according to the present invention comprises live attenuated RTX-toxin producing bacterium belonging to the species Pasteurella haemolytica. 
In a still even more preferred form of this embodiment, the antigens selected from other pathogenic microorganisms or viruses are chosen from the group of cattle pathogens, consisting of Bovine Rotavirus, Bovine Viral Diarrhoea virus, Parainfluenza type 3 virus, Bovine Paramyxovirus, Foot and Mouth Disease virus, Pasteurella multocida, Haemophilus somnus, Brucella abortus, Staphylococcus aureus, Streptococcus spp., Mycoplasma spp., and Bovine Respiratory Syncytial Virus.
There are several ways to store live organisms. Storage in a refrigerator is e.g. a well-known method. Also often used is storage at xe2x88x9270xc2x0 C. in a buffer containing glycerol. Bacteria can also be kept in liquid nitrogen. Freeze-drying is another way of conservation. Freeze-dried bacteria can be stored and kept viable for many years. Storage temperatures for freeze-dried bacteria may well be above zero degrees, without being detrimental to the viability.
Freeze-drying can be done according to all well-known standard freeze-drying procedures. Optional beneficial additives, such as e.g. skimmed milk, trehalose, gelatin or bovine serum albumin can be added in the freeze-drying process.
Therefore, in a preferred embodiment, the vaccine is in a freeze-dried form.
The invention also refers to the use of vaccines according to the present invention for the protection of susceptible animals against infection with bacteria of the family Pasteurellaceae.
In a preferred embodiment, vaccines according to the present invention are used for the protection of a susceptible animal against Actinobacillus pleuropneumoniae infection.
Also, the present invention relates to methods for the preparation of live attenuated RTX toxin producing bacteria of the family Pasteurellaceae.
Said methods comprise the introduction of a mutation in the gene encoding the RTX activator protein.
In a preferred embodiment of this method, the mutation to be introduced is a deletion in the RTX activator protein gene.
Finally, the present invention relates to methods for the preparation of a vaccine for the protection of animals against infection with an RTX toxin producing bacterium of the family Pasteurellaceae. One method comprises admixing bacteria according to the present invention with a pharmaceutically acceptable carrier as described above.